Placental tissue compositions

ABSTRACT

Compositions and associated methods of preparing placental tissue products/compositions are disclosed together with methods of utilizing the placental tissue composition for delivery of a therapeutic agent.

CROSS REFERENCE TO RELATED APPLICATIONS PRIORITY

This application is a U.S. national phase of International ApplicationPCT/US2018/016383 filed on Feb. 1, 2018, which claims priority to andbenefit of U.S. Provisional Patent Application Ser. No. 62/453,335,filed on Feb. 1, 2017. The entire contents of each of which isincorporated herein in its' entirety for all purposes.

FIELD

Disclosed herein are biological compositions, methods of makingcompositions, and methods of using compositions. In embodiments, acomposition is derived from placental membrane tissue.

BACKGROUND

The human placenta connects the fetus to the mother's uterine wall andis responsible for the protection and development of the fetuseffectively from conception to the time of birth.

SUMMARY

The present invention relates to therapeutic compositions comprisingplacental tissue and associated methods of preparing and using placentaltissue products/compositions. Placental tissue includes the placentaldisc and the amniotic sac. The amniotic sac comprises two primarylayers, the chorion and the amnion. In an embodiment, a composition ofthe present invention comprises ex-vivo placental tissue treated withhydrogen peroxide (H₂O₂). In another embodiment, a composition comprisesex-vivo placental tissue treated with peracetic acid (C₂H₄O₃). In someembodiments, a composition may comprise an ex-vivo amnion treated with aprocessing agent, wherein the processing agent comprises any one of anacid, a base, an anionic detergent, an ionic detergent, a Zwitterionicdetergent, an alcohol, an oxidizing agent, hypertonic solution,hypotonic solution, a solvent, a supercritical fluid, and combinationsthereof. In some embodiments, a composition may comprise an ex-vivoamnion treated with a processing agent, wherein the processed amnioncomprises an altered capacity to bind and/or release a pharmaceutical ortherapeutic agent. In embodiments, the placental tissue comprisesamnion. An amnion as described herein may comprise an amniotic membranein its entirety or a portion thereof. In embodiments the composition mayfurther comprise a therapeutic agent.

In certain embodiments, an amniotic membrane is processed to enhance itsfunction as a delivery device for therapeutic agents including smallmolecules, proteins, cytokines, growth factors, cells, acellularplacental products, and/or gene therapy agents. In an embodiment of thepresent invention, a method comprising processing an amnion in hydrogenperoxide to enhance the loading capacity of the amnion is provided. Inan embodiment, the present invention provides a method comprisingprocessing an amnion in peracetic acid to enhance the loading capacityof the amnion. In an embodiment, an amnion is loaded with therapeuticagents comprising small molecules, proteins, cytokines, growth factors,cells, gene therapy agents, and/or other therapeutic agents. In someembodiments, a method for enhancing a loading capacity of an amnion maycomprise contacting an amnion with a processing agent, wherein theprocessing agent comprises any one of an acid, a base, an anionicdetergent, an ionic detergent, a Zwitterionic detergent, an alcohol, anoxidizing agent, hypertonic solution, hypotonic solution, a solvent, asupercritical fluid, and combinations thereof.

In certain embodiments, compositions described herein may be used as awound covering. In some embodiments, compositions may be used for localdrug delivery. In some embodiments, the compositions may be used to aidin healing of burns, internal and external ulcers, surgical sites, nerveinjuries, or other lesions. In certain embodiments, a method fortreating a condition with an amnion may comprise loading a processedamnion with a therapeutic agent, wherein the therapeutic agent comprisesany one of small molecules, proteins, cytokines, growth factors, genetherapy agents, cells, and combinations thereof, and placing the loadedamnion at a treatment location of a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing depicting the layers of the placenta.

FIG. 2 is a micrograph of an amnion processed in hydrogen peroxide.

FIG. 3 is a micrograph of an amnion processed in peracetic acid.

FIG. 4 is a graph showing the loading of processed amnion after a 2-hoursoak in Dulbecco's phosphate buffered saline (DPBS).

FIG. 5 is a graph showing the loading of processed amnion after a24-hour soak in DPBS.

FIG. 6 is a graph showing the loading of protein in processed amnion.

FIG. 7 is a graph showing the cumulative percent release of protein fromthe processed and loaded amnion of FIG. 7.

FIG. 8 is a graph showing the cumulative percent release ofciprofloxacin with flux from processed amnion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions comprising placentaltissue and associated methods of preparing placental tissueproducts/compositions. The human placenta connects the fetus to themother's uterine wall and is responsible for the protection anddevelopment of the fetus effectively from conception to the time ofbirth. During pregnancy, the placenta helps the fetus develop byproviding nutrients and oxygen, by performing some immunity functions,and by releasing growth factors and cytokines, small proteins that actto direct cell migration.

During pregnancy, the placenta provides nutrients, growth factors, andcytokines to the fetus via the umbilical cord and amniotic fluid. Boththe fetus and placenta express antigens that are disparate from themother, yet avoid being rejected by the maternal immune system duringthe pregnancy. The residual cells, transport properties, and limitedimmune response of placental tissue help make it desirable and/oradvantageous to utilize placental tissue in medical applications.

Placental tissue may be typically collected after an elective Cesareansurgery. Placental tissue may also be collected after a normal vaginaldelivery. The tissue may be used unmodified in some applications.However, it would be advantageous to improve the performance ofplacental tissue for enhanced medical applications. For example, it isadvantageous and/or desirable to utilize placental tissue to delivertherapeutic agents for wound healing, osteoarthritis, pain managementand the like.

For purposes of describing certain embodiments of the present invention,reference is made herein to human placenta tissue. Embodiments of thepresent invention, however, are not limited to comprising naturallyoccurring human placenta tissue. Embodiments of the present inventionmay include, but are not limited to: natural or synthetic human placentatissue; natural or synthetic mammalian placenta tissue; natural orsynthetic placenta tissue from other animals, e.g., bovine, equine,porcine, ovis, capra, or camelid; and/or natural and/or syntheticcompositions having similar properties to placenta tissue. The placentatissue described herein comprises ex-vivo tissue, where ex-vivo relatesto use and treatment of the tissue outside the body of an organism.

Placenta tissue comprises the placental disc, the amniotic sac, theumbilical cord, its vessels, and Wharton's Jelly cushioning theumbilical cord vessels. The amniotic sac comprises the outer chorion andthe inner amnion. The chorion and amnion are separable membrane layers.

The innermost layer of the placental tissues comprises the amnion. Thismembrane covers the embryo when it first forms, then fills with amnioticfluid to protect the fetus during development. The amniotic fluidtransmits the placental-produced cytokines that orchestrate fetal growthand development from a single cell into a viable human. The amnioncomprises a shiny, tough, avascular tissue comprised of several layersof connective tissue and a single layer of non-ciliated cells as shownin FIG. 1. Histological evaluation indicates that the amnion comprises alayered membrane comprising an epithelial cellular layer, a thinreticular fibrous layer (basement membrane), a thick compact layer, afibroblast layer, and an intermediate spongy layer. The connectivetissue layers of the amnion contain various collagens and proteinsincluding collagen types I, III, IV, V, VI, as well as fibronectin,nidogen, laminin, and proteoglycans.

The maternal side of the amniotic sac comprises the chorion. The chorionmay be three to four times thicker than the amnion and substantiallycover the amnion. As shown in FIG. 1, the chorion comprises a reticularlayer, basement layer, and trophoblast layer. The trophoblast layer maybe adhered to the maternal decidua.

In vivo, the amnion may deliver between 200 and 500 mL of fluid per daybetween the placenta and amniotic fluid. Transfer of placental derivedcytokines and fluid across the amniotic membrane may occur primarily viaintramembranous transport either by diffusion through highly arborizedtight junctions between amniocytes or by trans-cellular vesicletransport, which has been shown to be bi-directional. Thus, every layerof the amnion may comprise suffused cytokines and fluid. The amnion mayfunction as a reservoir and delivery device for the cytokinessynthesized and stored by cyto- and syncytiotrophoblasts and Hofbauercells in the chorion.

Both the fetus and placenta may express antigens that are disparate fromthe mother, yet avoid being rejected by the maternal immune systemduring the pregnancy. The limited immuno response of the placentaltissue is believed to assist the placenta in avoiding fetal rejectionduring pregnancy.

The transport properties, residual cells, and limited immune response ofplacental tissue help make it desirable to utilize placental tissue inmedical applications. Placental tissue may be collected after anelective Cesarean surgery. Placental tissue may also be collected aftera normal vaginal delivery. Placental tissue may be obtained through FDAregistered tissue banks. The tissue may be used unmodified in someapplications. However, it would be advantageous to improve theperformance of the placental tissue for enhanced medical applications.

In embodiments, the present invention relates to placental tissueproducts and compositions. In some embodiments, a composition maycomprise an amnion and a processing agent. In some embodiments, theprocessing agent may comprise an oxidizing agent. In some embodiments,the oxidizing agent may comprise hydrogen peroxide and/or peraceticacid. In some embodiments, a composition may comprise an amnion and aprocessing agent, wherein the processing agent comprises any one of anacid, a base, an anionic detergent, an ionic detergent, a Zwitterionicdetergent, an alcohol, an oxidizing agent, hypertonic solution,hypotonic solution, a solvent, a supercritical fluid, and combinationsthereof. In some embodiments, a composition may comprise an ex-vivoamnion treated with a processing agent, wherein the processed amnioncomprises an altered capacity to bind and/or release a pharmaceutical ortherapeutic agent. In some embodiments, the processed amnion maycomprise an altered capacity to bind and/or release a pharmaceutical ortherapeutic agent. In some embodiments, the concentration of theprocessing agent may be up to 10% weight by volume of the amnion. Insome embodiments, the processing agent may comprise an anionicsurfactant solution, a strong basic solution, a strong acidic solution,a high concentration salt solution, and/or an oxidizing agent insolution with an alcohol. In some embodiments, the anionic surfactantsolution may comprise sodium dodecyl sulfate (SDS), Triton™ X-100,and/or Triton™ X-80.

In some embodiments, the loading capacity of the processed amnion isgreater than the loading capacity of the amnion that has not beenexposed to the processing agent. In some cases, a loading capacity ofthe processed amnion is at least 1.2 times greater than the loadingcapacity of the amnion that has not been exposed to the processingagent. In some cases, a loading capacity of the processed amnion is atleast five times greater than the loading capacity of the amnion thathas not been exposed to the processing agent.

The loading capacity of a processed amnion may be several times greaterthan the loading capacity of an unprocessed amnion. In some embodiments,the loading capacity of an unprocessed amnion may be up to about 0.1 mgprotein per mg of amnion. In some cases, the capacity of a processedamnion may be up to 3 mg protein per mg of amnion, for example, thecapacity may be about 2.9, about 2.8, about 2.7, about 2.6, about 2.5,about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2,about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about0.5, about 0.4, about 0.3, or about 0.2 mg protein per mg of amnion.

In some embodiments, the loading capacity of a processed amnion may beup to about 5 mg of therapeutic agent per mg of amnion. In someembodiments, the loading capacity of a processed amnion may be severaltimes greater than the loading capacity of an unprocessed amnion. Insome cases, the capacity of a processed amnion may be up to 7 mg oftherapeutic agent per mg of amnion. In some cases, the capacity of aprocessed amnion may be up to 10 mg of therapeutic agent per mg ofamnion, for example, the capacity may be about 10 mg, about 9.8 mg,about 9.6, about 9.4, about 9.2, about 9, about 8.8, about 8.6, about8.4, about 8.2, about 8, about 7.8, about 7.6, about 7.4, about 7.2,about 7, about 6.8, about 6.6, about 6.4, about 6.2, about 6, about 5.8,about 5.6, about 5.4, about 5.2, about 5, about 4.8, about 4.6, about4.4, about 4.2, or about 4 mg, about 3.8, about 3.6, about 3.4, about3.2, about 3, about 2.9, about 2.8, about 2.7, about 2.6, about 2.5,about 2.4, about 2.3, about 2.2, about 2.1, about 2.0, about 1.9, about1.8, about 1.7, about 1.6, about 1.5, about 1.4, about 1.3, about 1.2,about 1.1, about 1.0, about 0.9, about 0.8, about 0.7, about 0.6, about0.5, about 0.4, about 0.3, about 0.2, or about 0.1 mg therapeutic agentper mg of amnion.

In some embodiments, the composition comprises about 50% amnion andabout 50% processing agent. In some embodiments, the compositioncomprises up to about 75% processing agent. In other embodiments, thecomposition comprises at least about 5% processing agent. For example,the composition may comprise about 5%, about 7%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, or about 70% processing agent. Insome embodiments, the composition comprises at least 25% amnion. In someembodiments, the composition comprises up to about 95% of amnion. Forexample, the composition may comprise about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%amnion.

In some embodiments, at least one collagen-containing layer of theamnion may be expanded. As used herein, the expansion of a layer mayrelate to an increase in volume of a given layer of the amnion. In somecases, an epithelial layer of the amnion may be perforated. In certainembodiments, the amnion comprises a therapeutic agent may comprise smallmolecules, proteins, cytokines, growth factors, and/or cells.

In some embodiments, the amnion may be a sheet. The sheet may be 10 mmto 1 meter in width. In some embodiments, the amnion may be manipulatedto reduce the size of amnion particles or pieces. In some embodiments, asize of amnion may be between about 1 μm² and 10 mm². In otherembodiments, a size of amnion may be between about 10 mm² and 1000 cm².

In embodiments, the present invention relates to associated methods ofpreparing placental tissue products/compositions. In an embodiment, anamnion may be processed with a processing agent to enhance the capacityof the amnion. In some embodiments, the processing agent may sanitize orsterilize the amnion.

In some embodiments, a method for enhancing a loading capacity of anamnion may comprise contacting an amnion with a processing agent,wherein the processing agent comprises any one of an acid, a base, ananionic detergent, an ionic detergent, a Zwitterionic detergent, analcohol, an oxidizing agent, hypertonic solution, hypotonic solution, asolvent, a supercritical fluid, and combinations thereof. In certainembodiments, the contact step may comprise submerging, rinsing,perfusing, and/or spraying the amnion. In some cases, the method mayfurther comprise agitating and/or sonicating the amnion. In some cases,the method may further comprise pulverizing, grinding, chopping, ormicronizing the amnion. In some examples, the oxidizing agent maycomprise hydrogen peroxide and/or peracetic acid.

In some embodiments, the amnion may be processed by submersion, rinsing,spraying, perfusing, or otherwise treating the amnion with a processingagent. In some embodiments, the processing may be performed under staticconditions, with agitation, sonication, or otherwise. In someembodiments, the increased capacity of the amnion may permit the amnionto hold an increased amount of therapeutic agent for delivery.

In some embodiments, the composition may further comprise a therapeuticagent. In certain embodiments, the therapeutic agent may comprise anyone of small molecules, proteins, cytokines, growth factors, genetherapy agents, cells, and combinations thereof. In some cases, thetherapeutic agent may comprise antibiotics. In some cases, thetherapeutic agent may comprise analgesics. In some cases, thetherapeutic agent may comprise acellular placental products.

In an embodiment, an amnion may be processed with an oxidizing agent toenhance the capacity of the amnion. In some embodiments, a concentrationof the oxidizing agent may be up to 10% weight by volume of theprocessing solution. In some embodiments, the amnion may be processed ina solution comprising hydrogen peroxide. In some embodiments, thehydrogen peroxide concentration may be up to 10% weight by volume of theprocessing solution. The hydrogen peroxide may sanitize or sterilize theamnion. The hydrogen peroxide may expand the collagen-containing layersof the amnion. This expansion may permit an increased amount of solutionto enter the amnion thereby increasing the capacity of the amnion.

In another embodiment, the amnion may be processed in a solution ofperacetic acid. In some embodiments, the peracetic acid concentrationmay be up to 10% weight by volume of the processing solution. Theperacetic acid may sanitize or sterilize the amnion. The peracetic acidmay perforate or otherwise alter the epithelial layer of the amnion.This alteration may allow an increased amount of solution to enter theamnion, thereby increasing the capacity of the amnion.

In another embodiment, the amnion may be processed in an anionicsurfactant solution to expand the capacity of the amnion. Anionicsurfactants may include, but are not limited to Triton™ X-100, and/orTriton™ X-80. In some embodiments, the amnion may be processed in ionicdetergents, such as sodium dodecyl sulfate. In some embodiments, theamnion may be processed in a basic solution, such as sodium hydroxide,to expand the capacity of the amnion. In some embodiments, the amnionmay be processed in an acidic solution, such as acetic acid, to expandthe capacity of the amnion.

In other embodiments, the amnion may be processed in a hypertonicsolution, for example a solution of greater than 2% sodium chloride, toexpand the capacity of the amnion. In other embodiments, the amnion maybe processed in a hypotonic solution, for example a solution of lessthan 0.45% sodium chloride, to expand the capacity of the amnion.

In some embodiments, the amnion may be processed in a solvent to expandthe capacity of the amnion. Solvents may include, but are not limitedto, alcohols, acetone, and tri (n-butyl) phosphate (TnBP). In someembodiments, the amnion may be processed with Zwitterionic detergents toexpand the capacity of the amnion. Zwitterionic detergents include, butare not limited to, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS), sulfobetaine-10 (SB-10),and sulfobetaine-16 (SB-16).

In some embodiments, the amnion may be processed in solutions containinga combination of an acid, a base, an anionic detergent, an ionicdetergent, a Zwitterionic detergent, an alcohol, an oxidizing agent,hypertonic solution, hypotonic solution, and a solvent. For example, insome embodiments, the amnion may be processed in a solution containingSDS and acetone, TnBP and Triton™ X-100, CHAPS and sodium hydroxide, andperacetic acid in ethanol to expand the capacity of the amnion. In someembodiments, the amnion may be processed with a supercritical fluid.

In another embodiment, the amnion may be processed in a solution ofperacetic acid and then processed in a solution of hydrogen peroxide toexpand the capacity of the amnion. In some embodiments, the peraceticacid concentration of the initial processing may be up to 10% weight byvolume of the processing solution and the hydrogen peroxideconcentration of the subsequent processing may be up to 10% weight byvolume of the processing solution. In another embodiment, the amnion maybe processed in a solution of hydrogen peroxide and then processed in asolution of peracetic acid to expand the capacity of the amnion. In someembodiments, the hydrogen peroxide concentration of the initialprocessing may be up to 10% weight by volume of the processing solutionand the peracetic acid concentration of the subsequent processing may beup to 10% weight by volume of the processing solution.

In some embodiments, an amniotic membrane may be processed to enhanceits function as a delivery device for therapeutic agents including smallmolecules, proteins, cytokines, growth factors, and/or cells. Smallmolecules may include, but are not limited to: antibiotics, such aspenicillins, cephalosporins, cipros, erythromycins; analgesics, such asnarcotics, NSAIDs, acetomenophen; vasodilators; vasoconstrictors;neuroleptics; and/or other drugs that may be delivered orally,transdermally or intravenously. Proteins may include, but are notlimited to bone morphogenetic protein (BMP-1, BMP-2, BMP-7),insulin-like growth factors (IGF-1), basic fibroblast growth factors(bFGF), nerve growth factors (NGF), and/or vascular endothelial growthfactors (VEGF). In some embodiments, therapeutics may include acellularplacental products. Gene therapy agents may include nucleic acids,viruses, plasmids, viral vectors, plasmid DNA, linear DNA, mRNA, iRNA,and siRNA. Therapeutic agents that can be used in the describedcomposition can include, for example, those described and set forth inUS patent publication number US20060078604 by Kanios, Mantelle, andNguyen, which is incorporated herein by reference.

In an embodiment, an amnion is loaded with therapeutic agents comprisingsmall molecules, proteins, cytokines, growth factors, cells, and/or genetherapy agents. The processed amnion may be loaded with the therapeuticagent by submersion, rinsing, spraying, or otherwise treating with theprotein solution. The loading may be performed under static conditions,with agitation, sonication, or otherwise.

In some embodiments, the loaded amnion may be stored until time of use.In some embodiments, the amnion may be loaded at the time of use. Insome embodiments, the loaded amnion may be dehydrated and stored untiltime of use. In some embodiments, the amnion may be left in sheet formfor use. In some embodiments, the processing of the amnion may furthercomprise pulverizing, grinding, chopping, or micronizing.

In some embodiments, a processed amnion may have improved retention oftherapeutic agents, including small molecules, proteins, cytokines,growth factors, cells, acellular placental products, and gene therapyagents. In some embodiments, the processed amnion may have controlledrelease of therapeutic agents, including small molecules, proteins,cytokines, growth factors, cells, acellular placental products, and genetherapy agents.

To utilize the amnion in enhanced medical applications, in someembodiments, the amnion may be processed in advance of treatment andloaded just prior to patient application. In some embodiments, targetedmedical applications to utilize placental tissue to deliver therapeuticagents include but are not limited to wound healing, osteoarthritis,pain management and the like.

In some embodiments, compositions described herein may be used to aid inhealing of burns, internal and external ulcers, surgical sites, nerveinjuries, or other lesions. In certain embodiments, a composition may beused as a wound covering. In embodiments, the composition may be applieddirectly to a wound site. In some embodiments, the composition may be insheet form. In some embodiments, the composition may be applied as anintact sheet. In other embodiments, the composition may be applied as apaste. In other embodiments, the composition may be powder, pulverizedparticles, or micronized particles.

In some embodiments, compositions may be used for local drug delivery.In some embodiments, compositions may be used for delivery oftherapeutic agents including small molecules, proteins, cytokines,growth factors, cells, acellular placental products, and/or gene therapyagents. Small molecules may include, but are not limited to:antibiotics, such as penicillins, cephalosporins, cipros, erythromycins,analgesics, such as narcotics, NSAIDs, acetomenophen; vasodilators;vasoconstrictors; neuroleptics; and/or other drugs that may be deliveredorally, transdermally or intravenously. In some embodiments,therapeutics may include acellular placental products. Proteins mayinclude, but are not limited to BMP-1, BMP-2, BMP-7, IGF-1, bFGF, NGF,and/or VEGF. In some embodiments, therapeutics may include gene therapyagents. Gene therapy agents may include, but are not limited to, nucleicacids, viruses, plasmids, viral vectors, plasmid DNA, linear DNA, mRNA,iRNA, and siRNA.

In other embodiments, the composition may be injected. In someembodiments, the therapeutic-loaded processed amnion may be used tocontrol the bacterial growth at a wound site. In some embodiments, thetherapeutic-loaded processed amnion may be used to manage pain, e.g.,from an injury. In embodiments, the amnion may be loaded with a growthfactor and used to help speed healing. In some embodiments, the amnionmay be loaded with a bone growth factor, such as BMP-1, 2, or 7, andused to aid in fracture healing. In some embodiments, the amnion may beloaded with a vascular endothelial growth factor (VEGF) and used to aidin healing of damaged blood vessels. In some embodiments, the amnion maybe loaded with a nerve growth factor (NGF) to aid in the growth ofnerves. In some embodiments, the amnion may be loaded with muscle growthfactor, such as IGF-1 or bFGF, to aid in the growth of muscle.

Not intending to be bound by theory, it is believed that a compositionwith an increased loading capacity and amount of therapeutic retainedwithin the composition may be beneficial for drug delivery.

Also disclosed herein are methods for treating an a condition with anamnion. In some embodiments, a method for treating a condition with anamnion may comprise loading a processed amnion with a therapeutic agent,wherein the therapeutic agent comprises any one of small molecules,proteins, cytokines, growth factors, gene therapy agents, cells, andcombinations thereof and placing the loaded amnion at a treatmentlocation. In some embodiments, the processed amnion may release thetherapeutic agent at a controlled rate. In certain embodiments, theamnion may be placed by injecting, covering, packing, or enclosing theamnion at the treatment location. In some embodiments, the conditionstreated may comprise wound healing, osteoarthritis, and/or painmanagement.

Embodiments of the present invention, and their advantages areillustrated by the following Examples.

Example 1

An amnion was processed in a solution comprising hydrogen peroxide toexpand the capacity of the amnion. The transmission electron microscope(TEM) micrograph of the hydrogen peroxide-processed amnion shows anexpansion in the collagen-containing layers, as shown in FIG. 2. Thisexpansion may permit an increased amount of solution to enter theamnion, thereby increasing the capacity of the amnion.

An amnion was processed in a solution of peracetic acid to expand thecapacity of the amnion. The TEM micrograph of a peracetic acid-processedamnion shows an altered epithelial layer, as shown in FIG. 3. Theepithelial layer may be perforated by the processing with peraceticacid, which may allow an increased amount of solution to enter theamnion, thereby increasing the capacity of the amnion.

Example 2

Birth tissue (BT), including placentas and accompanying membranes andumbilical cord, were obtained from an FDA registered human tissueestablishment. BT were placed into 0.9% NaCl and kept between 1° C. and10° C. until arrival at the experimental laboratory. BT arrived within48 hours of the time of birth. Upon arrival at the laboratory, theamnion was separated from the placental tissue. The amnion was gentlycleaned in 0.9% NaCl to remove clots and debris. Solutions of 0.6%, 3%,and 6% hydrogen peroxide (H₂O₂) were prepared by diluting a 33% stocksolution of hydrogen peroxide with DPBS. Solutions of 0.5%, 1%, and 5%peracetic acid (PAA) were prepared from a 37% stock solution and dilutedin DBPS. The non-processed control amnion was stored in DPBS at 4° C.The amnion was divided into equivalent segments. The segments wereplaced in the respective solutions and stored with gentle agitation fora period of 2 or 24 hours at 4° C. At the end of the processing period,the amnion segments were removed from the solution, rinsed with DPBS andplaced on a smooth, non-adhering surface to air dry overnight at ambientconditions. The following day, equal size pieces of the amnion from eachsolution were divided, weighed, and then placed into DPBS. The amnionswere removed at periods of 2 and 24 hours and weighed to determine theamount of DPBS solution loaded into each processed amnion. The resultingdata is provided in FIGS. 4 and 5.

FIG. 4 shows the loading of Dulbecco's phosphate buffered saline (DPBS)in amnion after processing in various solutions to enhance the capacityof the amnion. DPBS closely approximates the physiological conditions ofpH and salinity of the human body. Amnion segments of equivalent sizewere processed in the expansion solution for a two-hour period (notshown) and a 24-hour period (shown in FIG. 4) and then soaked in asolution of DPBS for 2 hours and then weighed.

The data is presented as compared to data from a non-processed amnioncontrol that was maintained in DPBS. The segments of amnion processedfor 2 hours showed no improvement in loading capacity as compared to thenon-processed DPBS control segment. However, segments of amnionprocessed with the agent for 24 hours showed improved loading capacity.In particular, amnion treated with peracetic acid showed the largestincrease in capacity, especially at the highest concentrations (shown inFIG. 4). The 5% peracetic acid processed amnion more than doubled theamount loaded in the amnion as compared to the control DPBS. Thehydrogen peroxide-treated amnion exhibited similar behavior, with the 3%and 6% hydrogen peroxide solution processed amnions exhibiting anincreased loading as compared to the control. The Change Relative toDPBS is measured as [weight change of a sample]/[weight change ofcontrol], where weight change is [final weight−starting weight].

FIG. 5 shows the loading of DPBS in amnion after processing in varioussolutions to enhance the capacity of the amnion. Amnion segments ofequivalent size were processed in the expansion solution for a two-hourperiod (not shown) and a 24-hour period (shown in FIG. 5) and thensoaked in a solution of DPBS for 24 hours and then weighed. The data ispresented relative to a non-processed amnion control that was maintainedin DPBS. The segments of amnion processed for 2 hours showed noimprovement in loading capacity as compared to the non-processed DPBScontrol segment. However, most of the segments of amnion processed withthe agent demonstrated a loading capacity at least equivalent to DPBS,with several greatly exceeding the capacity of the control DPBS. Inparticular, hydrogen peroxide at 3% and 6% showed the largest increasein capacity (shown in FIG. 5). The 3% hydrogen peroxide demonstrated a51% increase while the 6% hydrogen peroxide solution processed amnionsdemonstrated an 81% increase in the level of DPBS loaded as compared tothe control. The 1% and 5% peracetic acid-processed amnion demonstrateda 3% and 4% increase respectively in loading capacity as compared to thecontrol DPBS. Not to be bound by theory, the non-processed control mayhave demonstrated increased loading due to the longer soak time (24hours as compared to 2 hours). The processed amnion also loaded anincreased amount of DPBS, but the increase was similar to thenon-processed amnion.

Example 3

Birth tissue (BT), including placentas and accompanying membranes andumbilical cord, were obtained from an FDA registered human tissueestablishment, BT were placed into 0.9% NaCl and kept between 1° C. and10° C. until arrival at the experimental laboratory. BT arrived within48 hours of the time of birth. Upon arrival at the laboratory, theamnion was separated from the placental tissue. The amnion was gentlycleaned in 0.9% NaCl to remove clots and debris. Solutions of 0.6%, 3%,and 6% H₂O₂ were prepared by diluting a 33% stock solution of H₂O₂ withDPBS. Solutions of 0.5%, 1%, and 5% PAA were prepared from a 37% stocksolution and diluted in DBPS. The non-processed control amnion wasstored in DPBS at 4° C. The amnion was divided into equivalent segments.One group of amnion was processed in DBPS, 0.6% H₂O₂ or 1% PAA for 24hours. Another group of amnion was processed with 1% PAA for 22 hoursand then processed with 0.6% H₂O₂ for 2 hours. A final group wasprocessed with 0.6% H₂O₂ for 2 hours followed by 1% PAA for 22 hours.All amnions were processed at 4° C. for a total of 24 hours. At the endof the processing period, the amnion segments were removed from thesolution, rinsed with DPBS and placed on a smooth, non-adhering surfaceto air-dry overnight at ambient conditions. The following day, equalsize pieces of the amnion from each solution were divided, weighed, andthen placed into a solution containing 7.5 mg/mL protein and allowed tosoak for 2 hours. The amnions were removed and the total protein loadedinto the amnion was quantified using a Bradford Assay. The amount ofprotein loaded on the amnion was determined by the difference betweenthe concentration of the starting protein solution (7.5 mg/mL) and theresulting concentration of the solution exposed to the amnion. Theresulting data from is provided in FIG. 6.

FIG. 6 shows the loading of proteins in amnion after processing invarious solutions to enhance the capacity of the amnion. Amnion segmentsof equivalent size were processed in the expansion solution for a24-hour period and then soaked in a protein solution for two hoursfollowed by measurement of the loaded protein concentration. For twosegments, processing solutions were used sequentially, with the fourthsegment processed first in the hydrogen peroxide solution for 2 hoursand then processed in the peracetic acid solution for 22 hours. Thefifth amnion segment, processed first in hydrogen peroxide for 2 hoursand then processed in peracetic acid for 22 hours, showed a significantincrease in protein loading capacity as compared to the non-processedDPBS control segment, at a nearly quadruple the loading. As shown inFIG. 6, the amount of protein loaded in the processed amnion wasgenerally equivalent to or greater than the control. Only the exclusiveperacetic acid processed amnion demonstrated a lower loading levelcompared to the control. Surprisingly, the amnion processed withhydrogen peroxide followed by peracetic acid demonstrated a significantincrease in the loading level as compared to the control.

Example 4

Birth tissue (BT), including placentas and accompanying membranes andumbilical cord, were obtained from an FDA registered human tissueestablishment. BT were placed into 0.9% NaCl and kept between 1° C. and10° C. until arrival at the experimental laboratory. BT arrived within48 hours of the time of birth. Upon arrival at the laboratory, theamnion was separated from the placental tissue. The amnion was gentlycleaned in 0.9% NaCl to remove clots and debris. Solutions of 0.6%, 3%,and 6% H₂O₂ were prepared by diluting a 33% stock solution of H₂O₂ withDPBS. Solutions of 0.5%, 1%, and 5% PAA were prepared from a 37% stocksolution and diluted in DBPS. The non-processed control amnion wasstored in DPBS at 4° C. The amnion was divided into equivalent segments.One group of amnion was processed in DBPS, 0.6% H₂O₂, or 1% PAA for 24hours. Another group of amnion was processed with 1% PAA for 22 hoursand then processed with 0.6% H₂O₂ for 2 hours. A final group wasprocessed with 0.6% H₂O₂ for 2 hours followed by 1% PAA for 2.2 hours.All amnions were processed at 4° C. for a total of 24 hours. At the endof the processing period, the amnion segments were removed from thesolution, rinsed with DPBS and placed on a smooth, non-adhering surfaceto air-dry overnight at ambient conditions. The following day, equalsize pieces of the amnion from each solution were divided, weighed, andthen placed into a solution containing 7.5 mg/mL protein and allowed tosoak for 2 hours. The amnions were removed and the release assay wasstarted. The amnions were covered with 200 μL DPBS. At 6, 12, and 24hours and 2, 3, 4, and 5 days, the DPBS solution was removed and theDPBS solution was frozen until the end of the experiment. Fresh DPBSsolution was placed on the amnion at each sample point. A Bradford Assaywas used to quantify the protein released for each sample. Thecumulative percent released is the [sum of the mass released up to andincluding the time point of interest]/[initial loaded mass]. Forexample, the cumulative percent released at time point 2 for a 6 unitstarting mass sample may be [2 units on time point 1+1 units on timepoint 2]/[6 unit], which gives a 50% cumulative release at time point 2.The resulting data from is provided in FIG. 7.

FIG. 7 shows the cumulative release of proteins from amnion loaded withprotein after processing in various solutions to enhance the capacity ofthe amnion. Amnion segments of equivalent size were processed in theexpansion solution for a 24-hour period and then soaked in a proteinsolution for two hours followed by measurement of the loaded proteinconcentration of the amnion. To determine the amount of proteinreleased, the loaded amnion was placed in a solution of DPBS and theamount of released proteins was measured. As with the protein loadingmethod, the processing solutions were combined for two segments, withthe segment processed in the peroxide solution for 2 hours and processedin the peracetic acid solution for 22 hours. All process treatmentsshowed fairly strong retention of protein over the time period observed,yielding not more than 30% cumulative percent release. Processing theamnion with hydrogen peroxide and then peracetic acid yielded the bestretention of 4.7%, performing nearly twice as well as the DPBS controlat 9.1%. The amnion processed with hydrogen peroxide and then peraceticacid retained 95% of the protein loaded in the processed amnion. Not tobe bound by theory, the retention of the protein within the amnioncoupled with the perforated epithelial layer and expanded collagenlayers, as shown in FIGS. 2 and 3, may provide a means to deliver atherapeutic agent to the target region for an extended period of time. Aloaded amnion that retains a therapeutic agent within the amnion anddemonstrates a low release rate may provide improved delivery of thetherapeutic agent in a medical application.

Example 5

Birth tissue (BT), including placentas and accompanying membranes andumbilical cord, were obtained from an FDA registered human tissueestablishment. BT were placed into 0.9% NaCl and kept between 1° C. and10° C. until arrival at the experimental laboratory. BT arrived within48 hours of the time of birth. Upon arrival at the laboratory, theamnion was separated from the placental tissue. The amnion was gentlycleaned in 0.9% NaCl to remove clots and debris. Solutions of 0.6%, 3%,and 6% H₂O₂ were prepared by diluting a 33% stock solution of H₂O₂ withDPBS. Solutions of 0.5%, 1%, and 5% PAA were prepared from a 37% stocksolution and diluted in DBPS. The non-processed control amnion wasstored in DPBS at 4° C. The amnion was divided into equivalent segments.The segments were placed in the respective solutions and stored withgentle mixing for a period of 24 hours at 4° C. At the end of theprocessing period, the amnion segments were removed from the solution,rinsed with DPBS and placed on a smooth, non-adhering surface to air-dryovernight at ambient conditions. The following day, equal size pieces ofthe amnion from each solution were divided, weighed, and then placedinto 100 μL of 20 mg/mL ciprofloxacin (or 2 mg per amnion segment). Theamnion were permitted to soak in the ciprofloxacin solution for 2 hoursand then 1 mL of DPBS was added to the tube containing the tissue andciprofloxacin solution to start the release portion of the assay. At 6,12, and 24 hours and 2, 3, 4, and 5 days, 200 μL of the solution on eachamnion segment was removed and the DPBS solution was frozen until thecompletion of the experiment and 200 μL of fresh DPBS was added back inthe solution on each amnion segment. A Bradford Assay was used toquantify the protein released for each sample. The cumulativeciprofloxacin released was quantified for each processing solutionsample by measuring the absorbance at 280 nM in a microplate reader. Theresulting data is provided in FIG. 8.

FIG. 8 shows the cumulative release of ciprofloxacin from amnion loadedwith ciprofloxacin after processing in various solutions to enhance thecapacity of the amnion. Ciprofloxacin is a small molecule anti-bacterialagent. Amnion segments of equivalent size were processed in theexpansion solution for a 24-hour period and then soaked in aciprofloxacin solution of known concentration for two hours. Todetermine the amount of ciprofloxacin released, the loaded amnion wasplaced in a solution of DPBS and the amount of released ciprofloxacinwas measured, while allowing for flux. All amnion segments processedwith peracetic acid performed better than the DPBS control anddemonstrated a lower cumulative percent of ciprofloxacin released. Theamnion processed with the lowest concentration of hydrogen peroxidedemonstrated the greatest level of improvement over the DPBS controlsegment with as much as 30% of the ciprofloxacin retained in theprocessed amnion. The DPBS control has a burst release of 85% of theciprofloxacin within the first 12 hours, but then slowed and ended with97% of the ciprofloxacin released. At two days, two treatments (0.6%peroxide and 1% peracetic acid) had yet to reach the 85% ciprofloxacinrelease that the control group showed in the first 12 hours. This lowrelease level may be desirable for drug delivery. Processing the amnionwith 0.6% hydrogen peroxide, 1% peracetic acid, or 5% peracetic acid allretained the ciprofloxacin for a longer period than the DPBS control.

Illustrations of Suitable Alloys, Products, and Methods

As used below, any reference to a series of illustrative composition ormethods is to be understood as a reference to each of those compositionsor methods disjunctively (e.g., “Illustrations 1-4” is to be understoodas “Illustration 1, 2, 3, or 4”).

Illustration 1 is a composition comprising an amnion treated with aprocessing agent, wherein the processing agent comprises any one of anacid, a base, an anionic detergent, an ionic detergent, a Zwitterionicdetergent, an alcohol, an oxidizing agent, hypertonic solution,hypotonic solution, a solvent, a supercritical fluid, and combinationsthereof.

Illustration 2 is the composition of any preceding or subsequentillustration, wherein the processed amnion comprises an altered capacityto bind and/or release a pharmaceutical or therapeutic agent.

Illustration 3 is the composition of any preceding or subsequentillustration, wherein the oxidizing agent comprises any one of hydrogenperoxide and peracetic acid.

Illustration 4 is the composition of any preceding or subsequentillustration, wherein a concentration of the processing agent may be upto 10% weight by volume of the amnion.

Illustration 5 is the composition of any preceding or subsequentillustration, wherein a loading capacity of the processed amnion is atleast 1.2 times greater than the loading capacity of the amnion that hasnot been exposed to the processing agent.

Illustration 6 is the composition of any preceding or subsequentillustration, wherein at least one collagen-containing layer of theamnion is expanded.

Illustration 7 is the composition of any preceding or subsequentillustration, wherein an epithelial layer of the amnion is perforated.

Illustration 8 is the composition of any preceding or subsequentillustration, wherein the amnion comprises a therapeutic agentcomprising small molecules, proteins, cytokines, growth factors, and/orcells.

Illustration 9 is the composition of any preceding or subsequentillustration, wherein the amnion is a sheet.

Illustration 10 is the composition of any preceding or subsequentillustration, wherein a size of amnion is between about 1 μm² and 10mm².

Illustration 11 is the composition of any preceding or subsequentillustration, wherein a size of amnion is between about 10 mm² and 1000cm².

Illustration 12 is the composition of any preceding or subsequentillustration, further comprising a therapeutic agent.

Illustration 13 is the composition of any preceding or subsequentillustration, wherein the therapeutic agent comprises any one of smallmolecules, proteins, cytokines, growth factors, gene therapy agents,cells, and combinations thereof.

Illustration 14 is the composition of any preceding or subsequentillustration, wherein the therapeutic agent comprises antibiotics.

Illustration 15 is the composition of any preceding or subsequentillustration, wherein the therapeutic agent comprises analgesics.

Illustration 16 is the composition of any preceding or subsequentillustration, wherein the therapeutic agent comprises acellularplacental products.

Illustration 17 is a method for enhancing a loading capacity of anamnion comprising contacting an amnion with a processing agent, whereinthe processing agent comprises any one of an acid, a base, an anionicdetergent, an ionic detergent, a Zwitterionic detergent, an alcohol, anoxidizing agent, hypertonic solution, hypotonic solution, a solvent, asupercritical fluid, and combinations thereof.

Illustration 18 is the method of any preceding or subsequentillustration, wherein the contact step comprises submerging, rinsing,perfusing, and/or spraying the amnion.

Illustration 19 is the method of any preceding or subsequentillustration, wherein the oxidizing agent comprises hydrogen peroxideand/or peracetic acid.

Illustration 20 is the method of any preceding or subsequentillustration, wherein a concentration of the oxidizing agent is up to10% weight by volume of the amnion.

Illustration 21 is the method of any preceding or subsequentillustration, further comprising agitating and/or sonicating the amnion.

Illustration 22 is the method of any preceding or subsequentillustration, further comprising pulverizing, grinding, chopping, ormicronizing the amnion.

Illustration 23 is a method for treating a condition with an amnioncomprising: loading a processed amnion with a therapeutic agent, whereinthe therapeutic agent comprises any one of small molecules, proteins,cytokines, growth factors, gene therapy agents, cells, and combinationsthereof and placing the loaded amnion at a treatment location of apatient.

Illustration 24 is the method of any preceding or subsequentillustration, wherein the processed amnion releases the therapeuticagent at a controlled rate.

Illustration 25 is the method of any preceding or subsequentillustration, wherein the placing comprises injecting, covering,packing, or enclosing the amnion at the treatment location.

Illustration 26 is the method of any preceding or subsequentillustration, wherein the conditions treated comprise wound healing,osteoarthritis, and/or pain management.

Various embodiments of the present invention are described. For purposesof explanation, specific configurations and details are set forth inorder to provide a thorough understanding of the embodiments. However,it will also be apparent to one skilled in the art that the presentinvention may be practiced without the specific details. Furthermore,well-known features may be omitted or simplified in order not to obscurethe embodiment being described.

While the invention is susceptible to various modifications andalternative constructions, certain illustrated embodiments thereof areshown in the drawings and described above in detail. It should beunderstood, however, that there is no intention to limit the inventionto the specific form or forms disclosed, but on the contrary, theintention is to cover all modifications, alternative constructions, andequivalents falling within the spirit and scope of the invention, asdefined in the appended claims.

The terms “comprising,” “having,” “including,” and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. The term connected is to beconstrued as partly or wholly contained within, attached to, or joinedtogether, even if there is something intervening. Recitation of rangesof values herein are merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range,unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individual recitedherein. All methods described herein can be performed in any suitableorder unless otherwise indicated or clearly contradicted by context. Theuse of any and all examples or exemplary language is intended merely tobetter illuminate embodiments of the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventor for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated or otherwise clearly contradicted by context.

What is claimed:
 1. A composition comprising an ex-vivo amnion treatedwith a solution comprising a processing agent, wherein the processingagent comprises hydrogen peroxide, peracetic acid, or combinationsthereof, wherein the amnion comprises at least one collagen-containinglayer and at least one epithelial layer, and wherein at least onecollagen-containing layer of the amnion is expanded.
 2. The compositionof claim 1, wherein a concentration of the processing agent in thesolution is up to 10 percent by volume.
 3. The composition of claim 1,wherein a loading capacity of the processed amnion is at least 1.2 timesgreater than the loading capacity of the amnion that has not beenexposed to the processing agent.
 4. The composition of claim 1, whereinan epithelial layer of the amnion is perforated.
 5. The composition ofclaim 1, wherein the amnion is a sheet.
 6. The composition of claim 1,wherein a size of the amnion is between about 1 μm² and 10 mm².
 7. Thecomposition of claim 1, wherein a size of the amnion is between about 10mm² and 1000 cm².
 8. The composition of claim 1, further comprising atherapeutic agent.
 9. The composition of claim 8, wherein thetherapeutic agent comprises proteins, cytokines, growth factors, orcombinations thereof.
 10. The composition of claim 8, wherein thetherapeutic agent comprises at least one of antibiotics, analgesics, oracellular placental products.
 11. A method for treating a condition at alocation in or on the body of a subject in need thereof with atherapeutically effective amount of the composition of claim 1comprising: a) loading the composition with a therapeutic agent, whereinthe therapeutic agent comprises any one of small molecules, proteins,cytokines, growth factors, gene therapy agents, cells, and combinationsthereof; b) placing the loaded composition at the treatment location ofthe patient; and c) contacting the treatment location with the loadedcomposition for a sufficient length of time to effect the treatment. 12.The method of claim 11, wherein the placing comprises injecting,covering, packing, or enclosing the composition at the treatmentlocation.
 13. The method of claim 11, wherein the conditions treatedcomprise wound healing, osteoarthritis, and/or pain management.